上泽充通大￥ Shanghai Jiao Tong University 1896 1920 1987 2006 Lecture 11-2 Genetic Engineering Chapter 11in BROCK BIOLOGY OF MICROORGANISMS GHAI J UNIVE Chen Feng School of Life Science and Technology, Shanghai Jiao Tong University http://micro.sjtu.edu.cn
1896 1920 1987 2006 Lecture 11-2 Genetic Engineering Chen Feng School of Life Science and Technology, Shanghai Jiao Tong University http://micro.sjtu.edu.cn Chapter 11 in BROCK BIOLOGY OF MICROORGANISMS
泽克通大学 上 Shanghai Jiao Tong University 1896 1920 1987 2006 II.Gene Cloning a。n。 am,.。A.1A00VCIr
1896 1920 1987 2006 II. Gene Cloning
11.5 Plasmids as Cloning Vectors Useful Properties of Plasmids as Cloning Vectors 1.Small size,easy to isolate and manipulate 2. Independent origin of replication,independently from direct chromosomal control?独立复制起点 3. Multiple copy number,making amplification of DNA possible:多拷贝数 4. Selectable markers,easy to detect and select the plasmid-containing clones:选择性标记 Chen Feng,Lecture of Microbiology
Chen Feng, Lecture of Microbiology 11.5 Plasmids as Cloning Vectors Useful Properties of Plasmids as Cloning Vectors 1. Small size, easy to isolate and manipulate 2. Independent origin of replication, independently from direct chromosomal control独立复制起点 3. Multiple copy number, making amplification of DNA possible多拷贝数 4. Selectable markers, easy to detect and select the plasmid-containing clones选择性标记
Plasmids pBR322 Clal 1.Relatively small,4361bp EcoRI Hind Ill Ampicillin BamHI resistance 2.20-30 copies per cell;can be Pst I amplified to 1000-3000 copies Sall per cell by inhibiting protein synthesis; direction of DNA 3.10kb foreign DNA can be replication from the origin inserted; Tetracycline 4.Single cleavage sites for resistance Origin of DNA various restriction enzymes; replication 5.Ampicillin resistance and tetracycline resistance. Chen Feng,Lecture of Microbiology
Chen Feng, Lecture of Microbiology Plasmids pBR322 direction of DNA replication from the origin 1.Relatively small, 4361bp 2.20-30 copies per cell; can be amplified to 1000-3000 copies per cell by inhibiting protein synthesis; 3. 10kb foreign DNA can be inserted; 4. Single cleavage sites for various restriction enzymes; 5. Ampicillin resistance and tetracycline resistance
Cloning with plasmid pBR322 BamHI site APR pBR322 TcR Insertion of foreign DNA BamHI sites causes inactivation of the BamHl digestion tetracycline resistance gene Foreign DNA BamHI (insertional inactivation) digestion Clal EcoRI Hind IIl DNA ligase Ampicillin BamHI Recyclized pBR322; Hybrid containing resistance no foreign DNA foreign DNA s PstI Sal I ApR TCR ApR Transformation of E.coli Transformants Transformants resistant to both resistant to ampicillin ampicillin and tetracycline but sensitive to tetracycline -Tetracycline resistance Figure 10-37 Brock Biology of Microorganisms 11/e Origin of DNA 2006 Pearson Prentice Hall,Inc. replication
Chen Feng, Lecture of Microbiology Cloning with plasmid pBR322 Insertion of foreign DNA causes inactivation of the tetracycline resistance gene (insertional inactivation)